Name: assessment of submitochondrial protein localization in budding yeast saccharomyces cerevisiae
Uploaded: 2021-07-19T15:00:00
Description: Watch this Scientific Journal Video about Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae at JoVE.com

我们感谢A. Tzagoloff博士（哥伦比亚大学）提供针对提交粒体标记蛋白Cyt的抗体。 b2、αKGD 和 Sco1。我们还感谢马里奥·恩里克·德巴罗斯博士（圣保罗大学）在制定本议定书期间所作的有益讨论和评论。
这项工作得到了圣保罗国家保护基金会（FAPESP）的研究资助（2013/07937-8）。
Fernando Gomes和Helena Turano也得到了FAPESP的支持，分别为2017/09443-3和2017/23839-7。Angélica Ramos也得到了Coordenação de Aperfeiçoamento de Pessoal de Nível Superior（CAPES）的支持。

1. 酵母细胞的生长
<ol><li>通过将一小部分细胞从-80°C甘油储备到YPD（1%酵母提取物，2%蛋白胨，2%葡萄糖）琼脂平板上来分离目标菌株的单个菌落。将板在30°C孵育2-3天。 注意：本方案中使用的酿酒酵母菌株来自BY4741（MATα;his3Δ1;leu2Δ0;met15Δ0;ura3δ0）。除营养标记基因外，该菌株不含任何缺失基因，也不携带任何质粒。因此，它...

<ol>
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N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Landscape+of+submitochondrial+protein+distribution.">Landscape of submitochondrial protein distribution.</a> Nature Communications. 8 (1), 00359 (2017).</li><li>Morgenstern, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Definition+of+a+high-confidence+mitochondrial+proteome+at+quantitative+scale.">Definition of a high-confidence mitochondrial proteome at quantitative scale.</a> Cell Reports. 19 (13), 2836-2852 (2017).</li><li>Zahedi, R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+the+yeast+mitochondrial+outer+membrane+reveals+accumulation+of+a+subclass+of+preproteins.">Proteomic analysis of the yeast mitochondrial outer membrane reveals accumulation of a subclass of preproteins.</a> Molecular Biology of the Cell. 17 (3), 1436-1450 (2006).</li><li>V&#246;gtle, F. -. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intermembrane+space+proteome+of+yeast+mitochondria.">Intermembrane space proteome of yeast mitochondria.</a> Molecular &amp; Cellular Proteomics. 11 (12), 1840-1852 (2012).</li><li>Gregg, C., Kyryakov, P., Titorenko, V. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+mitochondria+from+yeast+cells.">Purification of mitochondria from yeast cells.</a> Journal of Visualized Experiments: JoVE. (30), e1417 (2009).</li><li>Boldogh, I. R., Pon, L. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+intracellular+membranes+by+means+of+sodium+carbonate+treatment:+Application+to+endoplasmic+reticulum.">Isolation of intracellular membranes by means of sodium carbonate treatment: Application to endoplasmic reticulum.</a> Journal of Cell Biology. 93 (1), 97-102 (1982).</li><li>Glick, B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochromes+c1+and+b2+are+sorted+to+the+intermembrane+space+of+yeast+mitochondria+by+a+stop-transfer+mechanism.">Cytochromes c1 and b2 are sorted to the intermembrane space of yeast mitochondria by a stop-transfer mechanism.</a> Cell. 69 (5), 809-822 (1992).</li><li>Diekert, K., de Kroon, A. I., Kispal, G., Lill, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+subfractionation+of+mitochondria+from+the+yeast+Saccharomyces+cerevisiae.">Isolation and subfractionation of mitochondria from the yeast Saccharomyces cerevisiae.</a> Methods in Cell Biology. 65, 37-51 (2001).</li><li>Glick, B. 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这里介绍的方案已被成功使用并连续优化了很长时间，以确定提交软骨区室中的蛋白质定位13，14，18，21，22，23。该协议的可靠性和可重复性在很大程度上取决于线粒体制剂的纯度和完整性18。这两个要求都是通过在粗线粒体制剂?...

线粒体是真核细胞的基本细胞器，在生物能量学、细胞代谢和信号通路中起着至关重要的作用1。为了正确执行这些任务，线粒体依赖于一组独特的蛋白质和脂质，负责其结构和功能。萌芽 酵母酿酒酵母 已被广泛用作研究线粒体过程以及其他细胞器的模型系统2。线粒体基因组仅编码酵母中的八种蛋白质;绝大多数线粒体蛋白（~99%）由核基因编码，核基因在胞质核糖体上翻译，随后通过复杂的蛋白质导入机器导入到其正确的提交粒体区室3，4，5。因此，线粒体生物发生取决于核基因组和线粒体基因组的协调表达6，7。导致线粒体生物发生缺陷的基因突变与人类疾病有关8，9，10。
在过去的二十年中，针对高度纯化的线粒体的高通量蛋白质组学研究导致了酵母线粒体蛋白质组的全面表征，据估计酵母线粒体蛋白质组由至少900种蛋白质组成11，12，13，14。尽管这些研究提供了有价值的信息，但仍然需要四个线粒体亚室（即外膜（OM），膜间空间（IMS），内膜（IMS）和基质）中每种蛋白质的亚组织体定位。通过对两个较小的线粒体亚室（OM和IMS）进行蛋白质组学范围的研究，部分解决了这个问题15，16。最近，Vögtle和合作者通过生成酵母中递交软骨蛋白分布的高质量全球地图向前迈出了一大步。使用结合基于SILAC的定量质谱，不同的提交粒体分馏方案以及来自OM和IMS蛋白质组的数据集的综合方法，作者将818种蛋白质分配到四个线粒体亚室13。
尽管这些高通量蛋白质组学研究取得了进展，但我们对提交软骨蛋白质组组成的了解远未完成。事实上，在Vögtle及其合作者报告的986种定位于酵母线粒体中的蛋白质中，有168种不能被分配到四个提交粒体区室中的任何一个13。此外，作者没有提供有关预测外周附着于线粒体膜外围的蛋白质膜拓扑的信息。例如，不可能知道被指定为外周附着在内膜上的蛋白质是否面向基质或膜间空间。除了蛋白质组范围研究中这些缺失的数据外，关于大量线粒体蛋白的亚有机体定位，还有相互矛盾的信息。一个例子是蛋白酶Prd1，它在Saccharomyces基因组数据库（SGD）和Uniprot等通用数据库中被分配为膜间空间蛋白。令人惊讶的是，使用类似于这里描述的亚分案方案，Vögtle和合作者清楚地表明Prd1是一种真正的基质蛋白13。如上所述，需要阐明或重新评估许多线粒体蛋白的膜软骨定位。在这里，我们提供了一个简单可靠的方案来确定酵母线粒体蛋白的亚有机体定位。该协议由各种研究小组开发和优化，并已常规用于确定提交粒体定位以及许多线粒体蛋白的膜拓扑。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bacto Peptone</td>
			<td>BD</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Yeast extract</td>
			<td>BD</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>Beckman Ultra-Clear Centrifuge Tubes, 14 x 89 mm</td>
			<td>Beckman Coulter</td>
			<td>344059</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA fatty acid free)</td>
			<td>Sigma-Aldrich</td>
			<td>A7030</td>
			<td>Component of Homogenization buffer</td>
		</tr>
		<tr>
			<td>DL-Dithiothreitol</td>
			<td>Sigma-Aldrich</td>
			<td>43815</td>
			<td>Component of DDT buffer</td>
		</tr>
		<tr>
			<td>D-Sorbitol</td>
			<td>Sigma-Aldrich</td>
			<td>S1876</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>E9884</td>
			<td></td>
		</tr>
		<tr>
			<td>Galactose</td>
			<td>Sigma-Aldrich</td>
			<td>G0625</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma-Aldrich</td>
			<td>M1254</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethylsulfonyl fluoride (PMSF)</td>
			<td>Sigma-Aldrich</td>
			<td>P7626</td>
			<td>Used to inactivate proteinase K</td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>P3786</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>P0662</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S8501</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloroacetic acid (TCA)</td>
			<td>Sigma-Aldrich</td>
			<td>T6399</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma Base</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Zymolyase-20T from Arthrobacter luteus</td>
			<td>MP Biomedicals, Irvine, CA</td>
			<td>320921</td>
			<td>Used to lyse living yeast cell walls to produce spheroplast</td>
		</tr>
	</tbody>
</table>

assessment of submitochondrial protein localization in budding yeast saccharomyces cerevisiae

尽管最近在酵母线粒体蛋白质组的表征方面取得了进展，但大量蛋白质的膜软骨定位仍然难以捉摸。在这里，我们描述了一种确定酵母线粒体蛋白亚有机体定位的稳健而有效的方法，这被认为是线粒体蛋白功能阐明过程中的基本步骤。该方法涉及一个初始步骤，包括获得高纯度的完整线粒体。然后将这些线粒体制剂进行亚分割方案，包括低渗休克（肿胀）和与蛋白酶K（蛋白酶）孵育。在肿胀期间，外线粒体膜被选择性地破坏，允许蛋白酶K消化膜间空间室的蛋白质。同时，为了获得有关膜蛋白拓扑的信息，首先对线粒体制剂进行超声处理，然后用碳酸钠进行碱性提取。最后，离心后，通过SDS-PAGE和蛋白质印迹分析来自这些不同处理的沉淀和上清液组分。通过将目标蛋白质的蛋白质印迹图谱与已知标准进行比较，可以获得邻位点定位以及膜拓扑结构。

屈服粒体分馏方案的成功取决于获得高度纯化的完整线粒体。为此，在酵母细胞裂解过程中，细胞器的完整性必须几乎完全保持。这是通过使用细胞裂解方案实现的，该方案结合了细胞壁的酶促消化，然后使用Dounce均质机对质膜进行物理破坏。然后通过分类离心收集线粒体内容物。这种亚细胞分馏产生富集的线粒体部分，正如高水平的porin（Por1）的存在所证实的那样，这是一种线粒体标志物蛋白?...

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Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

尽管最近取得了进展，但许多酵母线粒体蛋白仍然具有完全未知的功能。该协议提供了一种简单可靠的方法来确定蛋白质的膜软骨定位，这对于阐明其分子功能至关重要。

萌芽酵母酿酒酵母中软骨蛋白定位的评估

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins ...

该协议旨在确定酵母线粒体蛋白的递交软骨定位，这被认为是线粒体蛋白功能阐明过程中的基本步骤。该协议适用于我们在不同生长条件下维持的酵母菌株。代表研究线粒体的强大研究工具。首先，将甘油储备中的条纹细胞储存在80°C下，放到YPD琼脂平板上，以从感兴趣的菌株中分离出单个菌落。并将板在30°C下孵育2至3天。通过将YPD琼脂平板中的2至3个单个菌落接种在含有10至30mlYPGal培养基的100ml锥形瓶中来制备发酵剂培养物。将烧瓶在30°C下孵育24小时，以180至200rpm的剧烈振荡。将发酵剂稀释到1 L新鲜YPGal培养基中，在600nm处的光密度小于0.1。在30°C下培养细胞，以180至200rpm剧烈振荡约12小时，直到光密度达到1至1.5。如文中所述，执行高度纯化的线粒体的分离。并按照制造商的说明，使用Bradford测定法测定高度纯化的线粒体制剂的蛋白质浓度。用冰冷的SEM缓冲液将蛋白质浓度调节至10mg / ml。将40升高度纯化的线粒体转移到四个1.5ml预冷和标记的微量离心管中。在第一管和第二管中加入360 l的SEM缓冲液。并在第三和第四管中加入360升EM缓冲液。使用移液方案，根据文本，在第二管和第四管中加入4L新鲜制备的蛋白酶K。轻轻混合所有试管后，在冰上孵育30分钟，偶尔混合。为了停止蛋白酶K活性，向所有四个管中加入4 L 200 mM PMSF。在4 C下以20， 000×g离心管30分钟，并在不干扰沉淀的情况下将上清液收集到新的1.5ml预冷标记微量离心管中。接下来，将沉淀重悬于400升冰冷的SEM缓冲液中。为了灭活可能的微量蛋白酶K，沉淀收集的上清液并用三氯乙酸重悬沉淀至终浓度为10%将管在冰上孵育10分钟。将三氯乙酸处理过的样品在4 C下以12， 000×g离心10分钟，并在除去上清液后，将沉淀重悬于200l样品缓冲液中。如果溴酚蓝变黄，加入1至5升1 M Tris基底，直至变蓝。向所有离心管中加入4升200 mM PMSF。将样品储存在80°C，直到通过SDS-PAGE和蛋白质印迹进一步分析。将200升高度纯化的线粒体转移到1.5ml预冷的微量离心管中。用冰冷的SEM缓冲液将线粒体稀释一倍。使用与小体积兼容的超声仪，在冰上超声处理线粒体3次，持续30秒。将样品在4 C下以100， 000×g离心30分钟，并将上清液收集在新的1.5ml预冷微量离心管中。将管标记为S"用于可溶性蛋白质部分后，将管保持在冰上。将上一步中的沉淀重悬于400升冰冷的SEM缓冲液中。将100l重悬沉淀转移到新的1.5ml预冷微量离心管中。将管子标记为SMP"，用于屈服粒体颗粒分数，并将管子保持在冰上。用新鲜制备的200mM碳酸钠将剩余的300l重悬沉淀稀释一倍。并将稀释后的样品在冰上孵育30分钟。然后，在4 C下以100，000×g离心样品30分钟，将上清液收集到新的1.5ml预冷微量离心管中。将样品标记为CS"以获得碳酸盐上清液部分，并将试管保持在冰上。将上一步中的沉淀重悬于400升冰冷的SEM缓冲液中。并将样品命名为CP"，用于碳酸盐沉淀馏分。用三氯乙酸沉淀所有样品至终浓度为10%，并将试管在冰上孵育10分钟。然后，在4 C下以12， 000×g离心样品10分钟，除去上清液后，将每个沉淀重悬于样品缓冲液中。如果样品缓冲液变为黄色，则加入1至5升1 M Tris碱，直到变为蓝色。向所有试管中加入1升200 mM PMSF，并储存在80°C，直到SDS-PAGE和蛋白质印迹进一步分析。在蔗糖密度梯度离心中纯化粗线粒体部分，并观察到其他细胞污染物的减少。线粒体到米托普斯特的转化通过沉淀中膜间空间蛋白细胞色素b2的消失来监测。伴随上清液中的外观。由于渗透性休克破坏外膜，观察到蛋白酶K介导的膜间蛋白Sco1的降解。通过保护细胞色素b2和Sco1防止沉淀级分中的蛋白酶K降解，证实了线粒体膜的外层完整性。类似地，基质可溶性蛋白KGD的保护证实了线粒体膜内部的完整性。Prx1蛋白质印迹图提示膜间空间和基质中的双线粒体定位。对线粒体进行超声处理和碳酸盐提取，以研究膜蛋白的拓扑结构。整体膜蛋白浇注保留在颗粒组分中。在上清液级分中检测到KGD。颗粒中KGD蛋白的检测是由于超声处理参数变化影响了SMP的形成。碳酸钠处理后，KGD和SMP级分被溶解并存在于上清液级分中。Prx1蛋白质印迹图提示与膜周边相关。为了提交软骨分馏方案的成功，重要的是通过向样品中添加PMSF来停止低渗肿胀后的蛋白酶K活性。位点提供有关膜软骨蛋白定位的信息。该协议还可用于检查线粒体制剂的完整性，这是线粒体亚室蛋白质组学研究期间的基础。

Research

JoVE Journal

Biochemistry

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

核糖体剖面对芽殖酵母中翻译的全基因组定量

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription ...

科研

生物化学

Evaluation of the Impact of Protein Aggregation on Cellular Oxidative Stress in Yeast

蛋白质聚集对酵母细胞氧化应激的影响评价

Protein misfolding and aggregation into amyloid conformations have been related to the onset and progression of several neurodegenerative diseases. ...

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

酵母 2-混合筛在批次中比较蛋白质相互作用

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

使用液色谱结合串联质谱法对糖皮质酶细胞脂质的定量分析

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

格拉菲克斯用于检测 糖精 的瞬态相互作用器

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, ...

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

贝克酵母糖精中线粒体基因组表达产品的标记和可视化

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A ...

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

使用液体色谱加上串联质谱法的 糖核 酸菌的定量元细胞学

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within ...

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

通过代谢标记和薄层色谱分析 糖浆 中中性脂质合成

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de ...

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

冷冻电子断层扫描 的糖核 酸的准备和冷冻-FIB微加工

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in ...

Isolation of Mitochondrial Contact Sites

An Improved Method to Isolate Mitochondrial Contact Sites

Unveiling Mitochondrial Contact Sites and Architectural Insights (Video) | JoVE

一种分离线粒体接触位点的改进方法

Mitochondria are present in virtually all eukaryotic cells and perform essential functions that go far beyond energy production, for instance, the ...